The specialized umbrella cells lining the mucosal surface of the urinary bladder form a barrier between the urine and the underlying muscle layers and vasculature. When this barrier function is disrupted, disease may ensue (e.g. in interstitial cystitis or bacterial infection). The barrier function of the uroepithelium depends on several factors including: formation of a polarized epithelium with tight junctions, presence of an apical plasma membrane highly impermeable to water and urea, and a surface layer of glycosaminoglycans. Understanding how barrier function is disrupted in disease requires that normal barrier function and development of the uroepithelium be defined. As such, the first aim of this proposal is to characterize the development of polarity and uroepithelial phenotype in a newly-established primary uroepithelial cell culture model. These cultures obtain high transepithelial resistance (up to 10,000 omegacm2), and have apical sodium channel activity. Like umbrella cells found in vivo, cultured umbrella cells have an asymmetric unit membrane and express the AE-31 antigen and uroplakins. In addition, two aspects of umbrella cell barrier function that are only poorly understood will be examined. These include the requirement that the umbrella cell accommodate large changes in bladder volume - likely to be mediated by fusiform vesicles that are inserted/removed from the apical membrane in response to filling and emptying of the bladder - as well as the ability of these cells to limit the amount of apical endocytosis and prevent endocytosed urine from reaching the underlying tissue. The hypothesis that fusiform vesicle exocytosis is regulated by stretch and will be modulated by stretch-sensitive channels, secondary messenger cascades, and SNARE fusion complexes will be examined in Aim II. The effects of stretch receptor antagonists on fusiform vesicle exocytosis will be examined, as will modulators of [Ca2+]i, cAMP production, protein kinase C activation, and SNARE fusion proteins. In Aim III the following hypotheses will be tested: (1) apical endocytosis will be inhibited during periods of bladder filling; (2) endocytosis will be stimulated following voiding; and (3) internalized urine will be primarily recycled to the apical pole of the cell or be delivered to lysosomes. A combination of biochemical and morphological tools will be used to characterize the endocytic and postendocytic traffic of stretched and unstretched primary cultured and freshly isolated uroepithelium. The proposed experiments will provide novel insights into normal uroepithelium development and barrier function.